A phenomenon that cytosine (C) located in the 5′ side of guanine (G) in 5′-CG-3′DNA part (hereinafter referred to as CpG site, or simply as CpG) present in the genome DNA sequence is methylated in mammals has been known. Methylation of CpG is considered to affect the gene expression. Especially, it is believed that CpG may significantly affect the gene expression, if a CpG-rich region (CpG island) is present within a gene promoter region.
Many CpG islands on chromosome are usually protected from methylation. However, once a CpG island within the promoter region is methylated for some reasons, the transcription of the gene is inhibited. For example, if a CpG island within the promoter region in a tumor suppressor gene in a human living body is abnormally methylated and the transcription of that tumor suppressor gene is inactivated, the cell proliferation gets out of control, which allows cell proliferative disorders such as cancers to progress.
Meanwhile, in the region outside the CpG island, cytosine in CpG is usually methylated. However, it has been reported that commonly methylated CpG's cytosines are not methylated in cancers and neoplasms.
With the recent progress of molecular biological approaches, the detection of the presence or absence of DNA methylation has enabled the early detection of cancers and tumors and the monitoring of medical treatment. For example, to rapidly detect methylation in nucleic acids containing CpG, methods for diagnosing cancers and the like through the use of PCR method are disclosed in JP 2000-511776 W (Patent Document No. 1), WO 02/38801 A1 (Patent Document No. 2) and Xiong Z, Laird P W. Nucleic Acids Res. 1997 Jun. 15; 25 (12): 2532-4 (Nonpatent Document No. 1). These methods put emphasis on specifically detecting methylated DNAs.
More particularly, following methods are disclosed: methods by preparing nucleic acid samples from various body fluids, tissues or cell lines, modifying unmethylated cytosine with, for example, bisulfite to be converted to uracil, then (1) amplifying them by PCR method (Methylation-Specific-PCR: MSP method) using a specific primer capable of distinguishing between methylated DNA and unmethylated DNA to detect methylated DNA (Patent Document No. 1) and (2) amplifying them by PCR method using a nonspecific primer incapable of distinguishing between methylated DNA and unmethylated DNA and treating with a restriction enzyme which recognizes difference in the base sequences within the PCR-amplified products to detect the presence or absence and/or the ratio of methylated DNA (Combined Bisulfite Restriction Analysis: COBRA method) (Nonpatent Document No. 1). Detecting the presence of methylated DNA within the base sequence of a particular gene through the use of these methods enables the early detection of cancers and tumors and the monitoring of medical treatment.
Further, a method of detecting methylated DNA and/or unmethylated DNA with a high sensitivity is disclosed (JP 2007-74950 A (Patent Document No. 3)). A method is disclosed wherein after a PCR amplification process, the amplified double-stranded DNA fragments are treated with exonuclease to obtain single-stranded DNA fragments which are detected by DNA microarray.
In MSP and COBRA methods, if a nucleic acid sample (DNA sample) is obtained not from tissues or cell lines but from various body fluids, the subject DNA is often not amplified due to a small amount of the contained DNA.
Usually, it has been known that the sensitivity (probability of obtaining amplified product by PCR method) and the specificity (probability of amplifying the target gene region only) of PCR method itself depend on the base sequence of the primer and the condition of PCR amplification reaction. In order to increase PCR sensitivity, the condition of the PCR amplification process should be loosened, which decreases the specificity of PCR.
MSP method uses primers each having specific sequence for methylated and unmethylated DNAs, respectively, in the amplification process. If the amount of template DNA is small, or if the purity is too low, for example, if various body fluids are used as specimens, the specificity is decreased because the condition of amplification process should be loosened. Therefore, it is hard to know whether the detected amplified product is really amplified from methylated DNA or unmethylated DNA only, and thereby problems arise. Further, there is a big difference between the sequences of a primer specific for unmethylated DNA and a primer specific for methylated DNA (for example, a primer for unmethylated DNA contains more As and Ts, while a primer for methylated DNA will contain more Cs and Gs), so that the amplification sensitivity and specificity may vary between methylated DNA and unmethylated DNA. Further, as the presence or absence of methylation and unmethylation is determined only by the presence or absence of PCR products, the error of the amplification process itself cannot be confirmed.
COBRA method has a problem of its low sensitivity toward methylated DNA. If various body fluids are used as specimens, the detection by COBRA method is likely to be impossible.    Patent Document No. 1: JP 2000-511776 W    Patent Document No. 2: WO 02/38801 A1    Patent Document No. 3: JP 2007-74950 A    Nonpatent Document No. 1: Xiong Z, Laird P W. Nucleic Acids Res. 1997 Jun. 15; 25 (12): 2532-4